T-Cell Lymphoma with Immune-Mediated Anemia and
Thrombocytopenia in a Horse
K.F. McGovern, K.M. Lascola, E. Davis, R.L. Fredrickson, and R. Tan
mild colic, and icterus.
Three days before the onset of clinical signs, the horse
received vaccinations for rabies, tetanus, West Nile
virus, Western equine encephalomyelitis, and Eastern
On examination the horse was quiet, alert, and
responsive. Heart rate was 54 beats/min, respiration
rate was 18 breaths/min, and temperature was 37.9°C.
Mucous membranes and sclera of the eyes were mark-
edly icteric and capillary refill time was 2 seconds.
Heart, lung, and abdominal auscultation were within
normal limits. Peripheral lymph nodes were not
enlarged and no abnormalities were detected on the
remainder of the physical examination.
Naso-gastric intubation revealed no net gastric
reflux. Abdominal ultrasonography revealed a small
area of moderately thickened (0.8 cm) ventral colon.
Thoracic ultrasound did not reveal abnormalities.
Rectal examination revealed splenomegaly but was
otherwise within normal limits. Abdominocentesis was
attempted but peritoneal fluid was not obtained.
Complete blood count (CBC) revealed anemia (PCV
16%; reference range 33–42%) and thrombocytopenia
(26,000/lL [reference range 100,000–600,000/lL]). Total
white blood cell count and differential were within nor-
mal limits. Cytological evaluation of peripheral blood
revealed rare nucleated red blood cells but otherwise
abnormalities were not detected. Serum biochemistry
abnormalities were limited to hyperglycemia (135 mg/
dL; reference range 60–107
bilirubinemia (10.9 mg/dL; reference range 0.5–2.5 mg/
dL). Direct and indirect bilirubin values were not mea-
sured. Total protein (TP) was 6.1g/dL (reference range
(275 g/dL; reference range 125–262 g/dL). Venous blood
gas analysisaidentified hyperlactatemia (7.1 mmol/L;
reference range <1 mmol/L) but was otherwise normal.
Prothrombin time and partial thromboplastin time were
normal. A direct Coombs test was negative.
13-year-old 480-kg mixed breed gelding was exam-
ined because of a 2-day history of anorexia, fever,
mg/dL) and hyper-
ELISA and agar gel immunodiffusion (Coggins test)
for equine infectious anemia and ELISA for Ana-
plasma phagocytophilum were negative.
Volume replacement included lactated Ringer’sb
(20 mL/kg bolus followed by 2 mL/kg/h IV mainte-
nance). Urine specific gravity (USG) after the initial
fluid bolus was 1.025 and dipstick analysis did not
reveal any abnormalities. Following the fluid bolus,
PCV was 13% and TP 5.6 g/dL. The horse was lethar-
gic and tachycardic (66 beats/min). Commercial fresh
frozen plasmacwas administered (4 mL/kg IV). Cross
matching was performed and 8 L whole blood was
administered from a donor horse. Treatment included
dexamethasoned(0.2 mg/kg, IV, q24h), trimethoprim
sulfamethoxazolee(TMS) (30 mg/kg, PO, q12h), and
sucralfatef(20 mg/kg, PO, q6h).
Because of the history of colic, feed was withheld for
the first 18 hours. No signs of colic or fever were noted.
The day after initial examination the horse was brighter
and vital parameters were within normal limits. PCV was
17% and blood lactate concentration was 2.2 mmol/L.
Gastroscopy was performed and did not reveal any
abnormalities. Feeding with small amounts of grass hay
was initiated. Intravenous administration of fluids was
discontinued 36 hours after initial examination.
On day 3, PCV was 20%. USG was 1.020 and fur-
ther urine dipstick analysis did not reveal any abnor-
malities. Mild hyperlactatemia persisted (2.8 mmol/L).
Whole blood was submittedgfor flow cytometry to
assess platelet and red blood cell surface associated
antibodies. For this test 10,000 red blood cells or
platelets are evaluated and the values given represent
the percentage of the total number of cells affected.
Platelet and red blood cell surface associated IgA anti-
body was normal (2% and <1%, reference range <4%
and <3%, respectively). Surface IgG antibody values
were 9% for platelets and 10% for red blood cells (ref-
erence range <4% and <3%, respectively). Surface
IgM antibody values were 8% and 4% (reference
range <4% and <3%). These findings are consistent
with immune-mediated thrombocytopenia (IMT) and
immune-mediated hemolytic anemia (IMHA) but do
not differentiate between primary and secondary causes.
On day 5, PCV was 24% and thrombocytopenia
was improving (68,000/lL). Hyperbilirubinemia per-
sisted (6.2 mg/dL) and rare nucleated red blood cells
were still noted in peripheral blood. Dexamethasone
was reduced to 0.1 mg/kg, IV q24h and antimicrobial
therapy was discontinued. An increase in respiratory
rate and effort was noted. Rectal examination, tho-
racic, and abdominal ultrasound remained unchanged.
Venous blood gas revealed worsening hyperlactatemia
From the College of Veterinary Medicine, University of Illinois,
Urbana, IL (McGovern, Lascola, Fredrickson, Tan);
Veterinary Medicine, Kansas State University, Manhattan, KS
Corresponding author: K. McGovern, College of Veterinary
Medicine, University of Illinois, 1008 W Hazelwood Drive,
Urbana, IL 61802; e-mail: firstname.lastname@example.org.
Submitted December 19, 2010; Revised March 24, 2011;
Accepted July 5, 2011.
Copyright © 2011 by the American College of Veterinary Internal
J Vet Intern Med 2011;25:1181–1185
(4.8 mmol/L). Intravenous administration of fluids
(lactated Ringer’s 2 mL/kg/h) was reinitiated and PCV
and TP closely monitored.
On day 6, fever of 103.3°F was noted, responsive to
phenylbutazone (2.2 mg/kg, IV). Over the next several
days, fevers of up to 104.7°F persisted but would
resolve for 12 hours after phenylbutazone administra-
tion. Hyperlactatemia persisted (3–6 mmol/L). Platelet
count and PCV remained stable at 70,000/lL and 22–
25% until day 8 when PCV declined (16%). Rare
nucleated red blood cells were still noted and the CBC
otherwise revealed no abnormalities. The horse was
intermittently dull but maintained a moderate appetite.
Treatment with dexamethasone, TMS, and sucralfate
Given the close association between vaccination
administration and the onset of IMHA and IMT, an
immune-mediated response to vaccination was consid-
ered likely. However, the poor response to corticoster-
oids along with recurrent fever and hyperlactatemia
led to a suspicion that IMHA and IMT were second-
ary to neoplasia. Because of the thickened ventral
colon, a rectal biopsy was performed and results did
not reveal any abnormalities. Abdominocentesis was
attempted again but remained unrewarding.
The horse’s condition deteriorated on day 11.
Marked tachycardia (70 beats/min), further decline in
PCV (13%), and hyperlactatemia (11.1 mmol/L) were
noted. A second whole blood transfusion (8 L) was
administered, dexamethasone was increased (0.2 mg/
kg, IV q24h), and treatment with oxytetracyclineh
(6.6 mg/kg, IV q12h) was initiated. Sucralfate contin-
ued to be given and administration of TMS was
discontinued. Lactate was 8–15 mmol/L and PCV was
9–14% for the next 48 hours. A further blood trans-
fusion was declined by the owner. Oxygen (10 L/min
intranasal) was administered.
A bone marrow biopsy was obtained from the tuber
coxae. Bone marrow cytology was nondiagnostic.
Histopathology from the same site revealed sparse
amounts of actual marrow consisting of collapsed
hemopoietic stroma with no active hemopoietic cells
present. Although regions were suspicious of myeo-
fibrosis, the sample was considered insufficient for a
Results of immunophenotyping of peripheral bloodg
were not consistent with neoplasia. Mild decreases in
CD4+T lymphocytes and mild increases in CD8+T
lymphocytes were noted and considered nonspecific.
Cell-cycle analysisgdetected populations of aneuploid
cells in peripheral blood which although inconclusive
could indicate the presence of circulating neoplastic cells.
Serum IgM levelsiwere decreased (<25 mg/dL [reference
range 67–543 mg/dL]) which suggested lymphoma.
On day 16 cytological analysis of peripheral blood
revealed the presence of rare lymphoblasts. The horse
was euthanized because of the high index of suspicion
of lymphoma and lack of response to treatment.
Gross necropsy findings revealed locally extensive,
red spongy areas in the diaphysis of both femurs. The
gastrosplenic, tracheobronchial, and cranial mediasti-
nal lymph nodes were mottled red and markedly
enlarged. Other lymph nodes were grossly unremark-
able. Several 2-cm arboriform depressions were noted
in the spleen. The remainder of the necropsy including
the colon (as an abnormality was suspected on the
ultrasound) was unremarkable.
Histopathology of left femur bone marrow revealed
approximately 60% of hemopoietic cells to consist of
a monomorphic population of neoplastic cells. The
neoplastic cells were round to ovoid with occasionally
cleaved central euchromatic nucleus, finely stippled
chromatin, and 1–3 prominent magenta nucleoli. There
was moderate anisocytosis, moderate anisokaryosis,
and karyomegaly. Approximately 45 mitotic figures
were present per 10 high power fields (400X). Approxi-
mately 40% of the hemopoietic cells in bone marrow
from one rib showed similar changes. Similar neoplas-
tic cells were seen in cranial mediastinal, gastrosplenic,
and tracheobronchial lymph nodes (Fig 1). In the
expanded by similar neoplastic cells, occasionally coa-
lescing with adjacent nodules. Immunohistochemical
stains were performed to determine the cell phenotype
which included CD3 which labels T cells and CD20,
CD79a, and BLA36 which label B cells. The neoplastic
cells labeled positive with CD3 and CD20 (Figs 2, 3)
and negative for CD79a and BLA36. A final diagnosis
of T-cell lymphoma was made.
Lymphoma is one of the most common neoplasms
in horses with prevalence estimated at 2.5%.1Clinical
signs are often varied and may include anemia and
Although well reported in the human literature, only
a single publication exists documenting IMHA and
IMT associated with neoplasia in horses.2In that
report, the diagnosis of IMHA in 3 horses was based
on clinical evidence of hemolysis and a positive
each have an approximately 2–3 erythrocyte wide, irregularly
round to ovoid, occasionally cleaved, central nucleus with finely
stippled chromatin and 1–3 prominent magenta nucleoli. There is
marked anisocytosis, marked anisokaryosis, and karyomegaly.
There are numerous bizarre mitotic figures. H&E stain. 100X.
Cranial mediastinal lymph node. The large neoplastic cells
1182McGovern et al
Coombs test. Although platelet factor 3 levels were
abnormal in 1 horse, the diagnosis of IMT was pre-
sumptive in the other 2 horses. Since that publication,
more sensitive diagnostic modalities have become
available to diagnose IMHA and IMT.
Immune-mediated hemolytic anemia and IMT are
caused by antibody-mediated destruction of red blood
cells and can occur independently or concurrently in
horses. IMHA occurs rarely as a primary disease.
IMHA can occur secondary to bacterial infection, neo-
plasia, inflammatory bowel disease, and purpura hem-
orrhagica in horses.3IMT in horses can be primary
(idiopathic) or secondary to drug administration,
neoplasia, or infection.4Flow cytometry to assess
platelet and red blood cell surface-associated antibod-
ies in this horse revealed increases in IgG and IgM
suggesting that hemolytic anemia and thrombocytope-
nia were caused by an immune-mediated process. Flow
cytometry techniques have been adapted for equine
and canine erythrocytes and platelets to detect mem-
brane-bound antibodies in cases of immune-mediated
anemia and thrombocytopenia.5Assays with isotype-
specific antibodies to equine immunoglobulins can elu-
cidate the class of cell-bound antibodies.6
The horse described in this report was negative for a
direct Coombs test. A negative Coombs test does not
rule out IMHA and it is estimated that approximately
30% of humans and canines with IMHA are negative
on a Coombs test.7Proposed explanations include lim-
itations in detection of globulins,2insufficient quanti-
ties of bound antibody,8or corticosteroid therapy.9
The horse in this case report had not received corticos-
teroids before blood collection for the Coombs test.
Autoagglutination in the absence of a positive direct
Coombs test may occur in the presence of low affinity
auto antibodies that eluted from erythrocytes when
Anemia is observed in approximately 1/3 of horses
with lymphoma and proposed causes include anemia
of chronic inflammatory disease (most common),
blood loss, IMHA hematopoietic dysplasia and leuko-
erythroblastic anemia.2In humans, lymphoma appears
to be the most common cancer associated with
immune-mediated destruction of red blood cells and
platelets (6–10% prevalence).11–15
Several theories have been proposed to explain the
association between neoplasia and immune-mediated
diseases. Neoplasia and immune-mediated disease can
be caused by a common immune system defect (inap-
propriate activation or inactivation of T cells).16Auto-
antibodies may be produced by the neoplastic process
itself.16In some T-cell lymphomas production of anti-
red cell antibodies can be part of a general stimulation
of the lymphoma.12Finally, cell molecular changes
might be responsible for the association between
conditions have been associated with oncogene over-
expression in lymphocytes.16
Fever is reported in approximately 50% of horses
with lymphoma and is thought to be caused by tumor
necrosis, pyrogens associated with neoplastic growth,
and secondary infections.17Lymphoma was a possible
cause of fever in this present horse; however, the
contribution of hemolysis is possible. Peripheral blood
cytological abnormalities were initially limited to the
presence of rare nucleated red blood cells. This is a
very unusual finding in horses as the equine bone mar-
row only rarely releases nucleated red blood cells into
the peripheral circulation in marked anemia.18,19In
other species, nucleated red blood cells can be seen
in peripheral blood with regenerative anemia, often
accompanied by findings such as reticulocytosis, poly-
chromasia, or macrocytosis. Other potential differen-
tials for the presence of nucleated red blood cells could
include myeloproliferative disorders, impaired splenic
function, or infiltrative marrow diseases such as
neoplasia.19Given the clinical and histopathological
findings in this horse and the lack of additional signs
and neoplasia as both
tic cells express diffuse, strong, membranous staining. CD3
diaminobenzidine substrate, hematoxylin counterstain. 40X.
Gastrosplenic lymph node. Majority of the large neoplas-
plastic cells express diffuse, strong, membranous staining. CD20
diaminobenzidine substrate, hematoxylin counterstain. 40X.
Gastrosplenic lymph node. The majority of the large neo-
Lymphosarcoma with IMHA and IMT 1183
of regenerative anemia in the peripheral blood or bone
marrow, the presence of nucleated red blood cells in
the peripheral circulation was considered most likely
secondary to neoplasia in the bone marrow.
Rare lymphoblasts were found in peripheral blood
on the day the horse was euthanized. The presence of
these cells suggests a secondary leukemic process which
has been reported in horses with lymphoma.20
Possible causes for the persistent hyperlactatemia
include tissue hypoxia secondary to inadequate tissue
perfusion, increased lactate release from the gastro-
worsened in this horse despite normovolemia and no
clinical evidence of cardiac disease, making impaired
tissue perfusion unlikely. Hyperlactatemia can occur
with severe anemia. Although the anemia may have
contributed to the hyperlactatemia, a direct associa-
tion between reductions in PCV and increases in
lactate concentration was not found making it unlikely
that this was the sole explanation for the increased
lactate. Although a small area of the ventral colon
appeared thickened on abdominal ultrasound, clinical
evidence of gastrointestinal disease was not found at
Studies have demonstrated that malignant transfor-
mation is associated with increases in anaerobic and
aerobic cellular lactate excretion. In a human study, in
all tumor types investigated, high concentrations of
lactate were correlated with a high incidence of distant
metastasis already in early stages of disease. Lactate
dehydrogenase (LDH) was found to be upregulated in
most tumors compared to surrounding normal tis-
sue.21In human studies of lymphoma, increased LDH
activity in serum are reported.15,22Similarly, lym-
phoma in dogs has been associated with increased serum
lactate concentration and LDH activity.23Although
speculative, it is possible that the hyperlactatemia noted
in this horse was associated with lymphoma.
Immunophenotyping and cell cycle analysis were
performed. Immunophenotyping (normal in this horse)
is performed with flow cytometry and may be normal
with equine lymphoma if the horse is not leukemic and
lymphocytes do not have abnormal protein expression.
Cell-cycle analysis is a research tool and is not rou-
tinely available. In this horse, the presence of aneu-
ploid cells in peripheral blood was further evidence of
neoplasia. Aneuploid cells are seen in lymphoma cases
as cells are in an abnormal state of cellular division.
IgM deficiencies can occur in horses with lym-
phoma. Deficiency can be secondary to a lack of pro-
duction of IgM if B-cell lines are obliterated (T-cell
lymphoma), or can occur because of inability of B cells
to synthesize IgM (B-cell lymphoma). The value of
decreased serum IgM values in equine lymphoma has
been evaluated.24At a lower limit of <23 mg/dL sensi-
tivity was poor (23%). However, the test had good spec-
ificity (88%) for supporting a diagnosis of lymphoma.
This T-cell lymphoma was concurrently positive with
CD20, a membranous antigen commonly associated
with B-cell phenotype as in humans.25Due to negative
CD79a/BLA36 stains, it is highly unlikely that this is
B-cell neoplasia and the unique aspect of CD20 label-
ing suggests an atypical subset of equine lymphoma.
aCritical Care Express, Nova Biomedical, Waltham, MA
bBaxter Healthcare Corporation, Deerfield, IL
cLake Immunogenics, Ontario, NY
dBimeda-MTC Animal Health Inc., Cambridge, ON
eAmneal Pharmaceuticals, Hauppauge, NY
fNostrum Laboratories, Kansas City, MO
gKansas State University Veterinary Diagnostic Laboratory,
hNorbrook Inc, Lenexa, KS
iWashington Animal Disease Diagnostic Laboratory, Pullman,
The authors express their gratitude to Dr. Ted Valli
and Dr. Luke Borst for their expertise and assistance
with this case report.
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Lymphosarcoma with IMHA and IMT1185